Accepted Manuscript Animal models of multiple endocrine neoplasia Tobias Wiedemann, Natalia S. Pellegata PII:

S0303-7207(15)30013-7

DOI:

10.1016/j.mce.2015.07.004

Reference:

MCE 9212

To appear in:

Molecular and Cellular Endocrinology

Received Date: 7 May 2015 Revised Date:

23 June 2015

Accepted Date: 3 July 2015

Please cite this article as: Wiedemann, T., Pellegata, N.S., Animal models of multiple endocrine neoplasia, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.07.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Animal models of multiple endocrine neoplasia Tobias Wiedemann and Natalia S. Pellegata, Institute of Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Ingolstaedter Landstrasse 1,

Multiple endocrine neoplasia (MEN) syndromes

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85764 Neuherberg, Germany.

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Multiple endocrine neoplasia (MEN) syndromes are autosomal dominant diseases with high penetrance characterized by proliferative lesions (usually hyperplasia or adenoma) arising in

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at least two endocrine tissues (Walls 2014). Four different MEN syndromes have been so far identified: MEN type 1 (MEN1), MEN2A (also referred to as MEN2), MEN2B (or MEN3) and MEN4, which have slightly varying tumor spectra and are caused by mutations in different genes (Thakker 2014). MEN1 associates with loss-of-function mutations in the MEN1 gene

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encoding the tumor suppressor menin (Lemos and Thakker 2008). The MEN2A and MEN2B syndromes are due to activating mutations in the proto-oncogene RET (Rearranged in Transfection) and are characterized by different phenotypic features of the affected patients

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(Walls 2014). MEN4 was the most recent addition to the family of the MEN syndromes. It was discovered less than 10 years ago thanks to studies of a rat strain that spontaneously

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develops multiple endocrine tumors (named MENX). These studies identified an inactivating mutation in the Cdkn1b gene, encoding the putative tumor suppressor p27, as the causative mutation of the rat syndrome (Pellegata et al. 2006). Subsequently, germline mutations in the human ortholog CDKN1B were also found in a subset of patients with a MEN-like phenotype and this led to the identification of MEN4. Small animal models have been instrumental in understanding important biochemical, physiological and pathological processes of cancer onset and spread in intact living organisms. Moreover, they have provided us with insight into gene function(s) and molecular 1

ACCEPTED MANUSCRIPT mechanisms of disease progression. We here review the currently available animal models of MEN syndromes and their impact on the elucidation of the pathophysiology of these diseases, with a special focus on the rat MENX syndrome that we have been characterizing. Animal models of MEN1

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MEN1 is a syndrome of tumors arising in the parathyroid, pancreatic islets, anterior pituitary and other endocrine organs. The most frequent MEN1-associated endocrinopathy, and often the first manifestation of the disease, is primary hyperparathyroidism (PHPT), which affects

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almost 100% of patients by the age of 50 yrs. PHPT is associated with the development of multiglandular parathyroid hyperplasia, which usually has a benign course and rarely to

parathyroid

gastroenteropancreatic

carcinoma

(GEP)

(Romei

et

al.

2012).

Additionally,

gastrinomas,

insulinomas,

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progresses

endocrine

tumors,

i.e.

glucagonomas, pancreatic polypeptidomas (PPomas), vaso-active intestinal peptidomas (VIPomas), nonfunctioning pancreatic tumors or carcinoid tumors, occur in approximately 3080% of patients (Calender et al. 1995; Trump et al. 1996; Marx 2005; Vierimaa et al. 2007;

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Thakker 2010). Approximately half of the GEP tumors are gastrinomas and they represent the main cause of morbidity and mortality given their tendency to metastasize. Pituitary tumors, including prolactinomas (most frequent), somatotropinomas (growth hormone-GH-

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producing), corticotropinomas and non-functioning adenomas, develop in 15-90% of MEN1 patients, and adrenocortical tumors in about 10% of cases. Less common MEN1-associated

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tumors include foregut carcinoids, lipomas, angiofibromas, angiomyolipomas, and spinal cord ependymomas (Schussheim et al. 2001; Jensen et al. 2008). MEN1 is caused by mutations in the MEN1 gene, which is localized on chromosome 11q13 and encodes a 610 amino acid protein referred to as menin, now proven to function as a tumor suppressor (Lemos and Thakker 2008). The MEN1 gene was cloned in 1997 (Chandrasekharappa et al. 1997) and the encoded protein was found to share no homology to any known protein and to possess no known functional domains, except for two classic nuclear localization signals. It was noted that MEN1 is widely expressed in normal tissues in 2

ACCEPTED MANUSCRIPT humans (Guru et al. 1999). Its mouse ortholog is also ubiquitously expressed, it has a genomic structure highly similar to that of the human gene, and the encoded proteins in both species share a 97% identity at the amino acid level (Stewart et al. 1998; Bassett et al. 1999). Over the years, information about the function of menin has been gathered through

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the identification of its various binding partners in biochemical studies, and through functional in vitro studies. In addition, animal models with defective menin function have further established the critical role of this protein in endocrine tissues. Indeed, various conventional (=constitutive) (Crabtree et al. 2001; Bertolino et al. 2003d; Loffler et al. 2007; Harding et al.

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2009) and tissue-specific (=conditional) (Bertolino et al. 2003h; Crabtree et al. 2003; Libutti et al. 2003; Biondi et al. 2004; Shen et al. 2009; Lu et al. 2010; Shen et al. 2010; Veniaminova

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et al. 2012) Men1 gene knockout mouse models have been generated and characterized, and have significantly contributed to our understanding of menin’s function in endocrine tumorigenesis.

It was first observed that constitutive homozygous deletion of Men1 in mice causes severe

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developmental defects and embryonic lethality at days E10.5 - E14.5. Thus, the studies of these mouse lines focused on heterozygous deficient animals (Crabtree et al. 2001; Bertolino et al. 2003a; Lemos et al. 2009).

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Crabtree and colleagues (Crabtree et al. 2001) were the first to generate a conventional Men1 knockout model where mice heterozygous for the introduced mutation (Men1∆N3-8/+;

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deletion of exons 3-8) phenotypically resembled patients affected by the MEN1 syndrome. These Men1-deficient mice developed pancreatic islet hyperplasia at 9 months of age, followed by insulin-producing islet cell tumors (insulinomas; frequency 28% by 22 months and younger). These hyperplastic or neoplastic lesions in the pancreas were associated with increased insulin levels, as seen in MEN1 patients with insulinomas (Thakker et al. 2012). Men1∆N3-8/+ mice showed parathyroid adenomas as early as 9 months of age (24%), and by the age of 16 months they also presented with pituitary tumors (26%), consisting mainly of prolactinomas. Adrenocortical tumors (20%), and, more rarely, gastric neuroendocrine 3

ACCEPTED MANUSCRIPT tumors and thyroid abnormalities were also detected in these mice. Gastrinomas, the predominant MEN1-associated extrapancreatic tumor type, and pancreatic glucagonomas, could not be detected in this animal model. Surprisingly, total calcium levels were unchanged in Men1∆N3-8/+ mice when compared with wild-type littermates. Hormonal disturbances

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commonly seen in humans, e.g. changes in parathyroid hormone (PTH) levels, were not assessed (Crabtree et al. 2001). Importantly, all tumors in Men1∆N3-8/+ mice showed loss-ofheterozygosity (LOH) of the wild-type Men1 allele, thereby further supporting the notion that Men1 is a tumor suppressor gene in endocrine cells. These findings are in agreement with

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the lack of menin expression seen in the tumors of MEN1 patients (Lemos and Thakker 2008).

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A second constitutive Men1 defective mouse model (Men1+/T; deletion of Exon 3) was generated by Bertolino and colleagues (Bertolino et al. 2003d). The heterozygous Men1+/T mice developed pancreatic islet hyperplasia at 8-12 months of age (65% of mice), which progressed to adenoma (insulinomas, glucagonomas and mixed hormone-producing islet

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tumors) and further to carcinoma. After 18 months, 46% of Men1+/T mice presented with adrenocortical adenomas or carcinomas. Parathyroid adenomas were detected in 41% of heterozygous mice in the 13-18 months age range, but they did not associate to increased

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PTH levels. Pituitary adenomas were detected in 19% of Men1+/T mice between 13 and 18 months of age, and their incidence increased to 37% in mice older than 18 months. Female

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Men1+/T mice were more prone to pituitary adenomas (79%) than males. In these mice also thyroid hyperplasia and tumors were identified at low frequency (6,5%). Interestingly, in addition to tumors belonging to the MEN1 spectrum, Men1+/T mice also revealed a high incidence of gonadal tumors of endocrine origin. Indeed, Leydig cell tumors of the testis were observed in up to 88% of male Men1+/T mice older than 18 months, and ovarian sex-cord stromal tumors in 50% of the females in the same age group. These neoplasias were not detected in Men1∆N3-8/+ mice (Crabtree et al. 2001). Moreover, always in contrast to the findings of Men1∆N3-8/+ mice, GEP, parathyroid and adrenal tumors in Men1+/T mice frequently 4

ACCEPTED MANUSCRIPT progressed to carcinoma (Bertolino et al. 2003d). The assessment of circulating hormone levels (i.e. PTH, insulin) in Men1+/T mice completed their phenotypic characterization and established additional similarities with the human MEN1 syndrome. A further conventional Men1 knockout mouse strain was generated by Loffler and colleagues

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(Loffler et al. 2007). In these genetically engineered mice, exon 2 of Men1, including the translation start site, was deleted (Loffler et al. 2007). Heterozygous Men1+/- mice share several phenotypic features with MEN1 patients in that they were found to develop pancreatic islet tumors in up to 90% of mice 18 months and older, pituitary adenomas (70%

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of females, 31% of males older than 18 months; mainly prolactinomas), hyperplasia or adenomas of the parathyroid glands (17% of mice), adrenocortical tumors (8%), as well as

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hyperplasia or tumor in testis (30%) and ovaries (16%). Interestingly, thyroid follicular epithelial hyperplasia occurred quite frequently in Men1+/- mice (>20% of mice older than 18 months). Similar to the model of Crabtree at al (Crabtree et al. 2001), progression of the various endocrine tumors to invasive carcinomas or metastases was not a feature of these

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mice. Noteworthy, while LOH was observed at all tumor sites, in testicular tumors it only reached the frequency of 36%.

Harding and colleagues (Harding et al. 2009) have established a Men1+/- mouse model

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(deletion of exon 1 and 2, including the ATG initiation codon) where the transgenic animals developed a very broad spectrum of endocrine tumors including pancreatic islet adenomas

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containing insulin and glucagon (60% of mice older than 12 months), parathyroid adenomas or focal hyperplasia, anterior pituitary adenomas (mainly GH and prolactin positive) with a gender bias, thyroid follicular and C cell tumors (11.5%), adrenocortical tumors (which occurred only in male Men1+/- mice), gonadal tumors (Leydig cell tumors in males and sexcord tumors in females) and lipomatous tumors (Harding et al. 2009). Gastrinomas were not identified in these mice. Circulating hormones and tumor biomarkers were thoroughly analyzed in affected Men1+/- mice and showed the occurrence of hypercalcaemia and hypophosphataemia in association with parathyroid hyperplasia and adenomas, albeit the 5

ACCEPTED MANUSCRIPT serum levels of PTH were within the normal range. In addition, Harding et al. (Harding et al. 2009) could demonstrate somatostatin receptor type 2 (Sstr2) and vascular endothelial growth factor-A (VEGF-A) expression in pancreatic islet cell tumors and anterior pituitary tumors of Men1+/- mice, similar to what has been reported for these tumor entities in man

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(Scoazec 2013; Theodoropoulou and Stalla 2013). These molecules could represent useful targets for the treatment of these tumors with somatostatin analogs or anti-angiogenic drugs. At the molecular level, LOH in the neoplasms of Men1+/- mice was detected in pancreatic islets tumors (83% of cases), in pituitary adenomas (100%) and in adrenocortical tumors

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(67%).

For the sake of completeness, it is should be pointed out that, in addition to the above

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described models, Scacheri et al. generated a conventional Men1 knockout mouse model where exons 2-4 of the gene were replaced by a phosphoglycerate kinase-neomycin (PGKneo) cassette (Scacheri et al. 2001). Unexpectedly, the monoallelic deletion of Men1 in these mice resulted in lethality.

Analysis of the heterozygous mice revealed late gestational

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lethality with some embryos showing omphalocele. The authors hypothesized that this severe phenotype was caused by the presence of the PGK-neo cassette rather than to the deletion of Men1. They proposed that the aberrant Men1 transcript generated from the

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transgenic allele has a toxic effect at the RNA level, or exerts a dominant negative effect by producing a truncated protein product.

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In summary, the conventional knockout models here presented represent relatively faithful models of human MEN1 as they recapitulate the main features of the disease (Table 1). The slight discrepancies in tumor spectrum and tumor frequency among these models are likely explained by the type of mutation introduced in the different mouse lines, or by the genetic background of the mice. Although the tumor spectrum of the heterozygous transgenic mouse lines generated closely resembles that of the human MEN1 syndrome, the incidence of the various tumor types is different in the two species. Indeed, while the most penetrant and often the first phenotypic 6

ACCEPTED MANUSCRIPT feature of MEN1 is PHPT associated with tumors of the parathyroid, occurring in almost 100% of older patients, in mice the first and most frequent tumor manifestation is pancreatic hyperplasia and tumors. Moreover, the mouse parathyroid neoplasias did not necessarily associate with altered level of PTH even if their phenotype was compatible with hormone

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hyperactivity. Extra-pancreatic GEP tumors only develop in some mouse lines and at variable frequency, while in MEN1 patients they are significantly more often present. Interestingly, there is a female prevalence in the incidence of pituitary adenomas associated with defective menin

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function both in mice and man. In contrast, the gender bias associated with the adrenocortical tumors in Men1+/-mice (seen only in males) has not been reported in MEN1

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patients. Occurring at high frequency in the above mouse models but not in the human syndrome are gonadal endocrine tumors, attesting to a species-specific sensitivity of these tissues to impaired menin function. In several mouse lines, Men1 monoallelic deletion predisposes to thyroid tumors. Thyroid adenomas and carcinomas have been reported to

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occur in 25% of MEN1 patients (Scarsbrook et al. 2006). However, given the high incidence of these tumors in the general population, it has been debated whether they are associated with the syndrome or may be incidental tumors. Studies of heterozygous Men1-deficient

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mice, showing that they develop thyroid tumors which have lost both wild-type Men1 allele and menin expression, support the hypothesis that these tumors develop as a consequence

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of their genetic defect. However, a recent report assessing the prevalence of thyroid tumors in patients with or without MEN1 suggests that these neoplasms may not be a feature of the MEN1 syndrome in man (Lodewijk et al. 2015). At the genetic level, these animal models have demonstrated that loss of both Men1 alleles in mice is embryonic lethal, thereby indirectly indicating that menin plays an important role in development. Moreover, they have shown that loss of one allele of Men1 predisposes mice to hyperplasia, but that the progression to tumors requires the loss of the wild-type allele. Indeed, LOH and lack of menin expression was observed in pancreatic, pituitary and other 7

ACCEPTED MANUSCRIPT tumors developing in the heterozygous Men1-deficient mice, consistent with a tumor suppressor role of the gene, and in agreement with findings in MEN1-associated tumors in patients (Larsson et al. 1988; Lubensky et al. 1996; Harding et al. 2009). In contrast, LOH was less frequently seen in the testicular tumors observed in heterozygous Men1+/- male

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mice (Loffler et al. 2007), indicating that a different molecular mechanism ensures tumor development in this tissue, which maybe involves genetic defects in genes other than Men1. To study in detail the consequences of homozygous deletion of Men1, additional mouse models have been generated with tissue-specific deletion of the gene. In the majority of

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these models, the gene was conditionally knocked-out in pancreatic endocrine cells (reviewed by R. Yu in this issue). There was however a model of hyperparathyroidism

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associated with the deletion of Men1 specifically in parathyroid cells (Libutti et al. 2003). In this model, no phenotype was reported for the heterozygous mice while the homozygously deleted mice showed histological changes in their parathyroid glands compatible with neoplastic transformation starting from 9 months of age (80% of mice). Together with

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parathyroid neoplasia, consistently elevated serum calcium levels were found in these mice. Given that gastrinomas occur frequently in MEN1 patients (mainly affecting the duodenum) but rarely in the conventional knockout models, Veniaminova and colleagues (Veniaminova

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et al. 2012) deleted Men1 in the epithelial lineages of the small and large intestines, as well as in the stomach antrum, by taking advantage of mice expressing the Cre recombinase from

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a modified villin transgene. Defective menin function in these mice elicited increased proliferation of the gastrin-producing G cells in duodenum and stomach antrum, antral G cell hyperplasia and hypergastrinemia, but was not sufficient to cause gastrinomas. These findings are in agreement with those of the constitutive Men1 knockout model of Crabtree et al. (Crabtree et al. 2001) but in contrast to the model described by Bertolino et al. (Bertolino et al. 2003d), where the loss of menin was associated to gastrinoma formation. Overall, these conditional knockout lines have shown that they can be useful to study tissuespecific effects of loss of menin function (Table 1). 8

ACCEPTED MANUSCRIPT Animal models of MEN2A and MEN2B In the various MEN2 syndrome variants, medullary thyroid cancer (MTC), which derives from calcitonin-producing C-cells, is the most frequent phenotypic feature and the first neoplastic manifestation in affected patients. Thyroid tumors are usually bilateral, multicentric and

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associated with C-cell hyperplasia (CCH). The MEN2A (MEN2) syndrome is the most common variant and is characterized by MTC (90% of cases) in combination with pheochromocytoma (40 - 50% of cases) and multiple tumors of the parathyroid glands (1020% of cases). The MEN2B (MEN3) variant is characterized by MTC, pheochromocytoma (in

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50% of cases), marfanoid habitus, mucosal and digestive ganglioneuromatosis. MEN2B is the least common (about 5% of all cases) but the most aggressive variant of the MEN2

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syndromes (Vierimaa et al. 2007; Thakker 2010). A milder variant of MEN2 exists where patients develop MTC but not the other neoplastic features, and it is therefore referred to as familiar medullary thyroid cancer (FMTC) (Vierimaa et al. 2007; Thakker 2010). The MEN2A and MEN2B syndrome variants are caused by germline mutations in the RET

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gene. The human RET gene maps to chromosome 10q11.2 and is composed of 21 exons. All MEN-associated mutations so far identified are gain-of-function mutations and convert the RET receptor tyrosine kinase into a dominantly-acting transforming protein, able to transmit

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signals of cell growth and survival independently from the binding to its physiological ligand. In MEN2A, the position of the mutation in RET is strongly associated with the disease

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phenotype (genotype-phenotype correlation) with regard to age of onset and aggressiveness of MTC, as well as to the presence or absence of other endocrine tumors. To date, several germline mutations of the RET proto-oncogene associated with MEN2A and FMTC have been described in humans (Raue and Frank-Raue 2010). The most frequent mutation in MEN2A patients is a missense mutation of codon 634 (C634R) in exon 11 (Donis-Keller et al. 1993; Mulligan et al. 1995; Eng et al. 1996; Frank-Raue et al. 1996). In MEN2B, a single point mutation at codon 918 (M918T) in exon 16 is responsible for approximately 95% of

9

ACCEPTED MANUSCRIPT cases (Vasen et al. 1992; Carlson et al. 1994; Hofstra et al. 1994; Santoro et al. 1995; Eng et al. 1996). Over the last two decades, a few genetically engineered mouse lines have been generated to model the different MEN2 syndrome variants (Table 1). A conventional knockout mouse

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model for the MEN2B syndrome (RetMEN2B/+, RetMEN2B/MEN2B) variant was created by SmithHicks and colleagues (Smith-Hicks et al. 2000) by introducing into the murine Ret gene the M919T codon alteration, which corresponds to the M918T mutation in man. The heterozygous transgenic mice (RetMEN2B/+) developed thyroid CCH (in 31% of mice at the age

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of 4-7 months, in 41% at 8-12 months), adrenal chromaffin cell hyperplasia (16-17% at 4-12 months of age) and pheochromocytoma (2% at 8-12 months of age). Homozygous mutant

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mice (RetMEN2B/MEN2B) displayed more severe thyroid lesions with respect to RetMEN2B/+ mice: CCH with a 86% frequency at the age of 6-10 months, which however did not progress to MTC, and pheochromocytomas (100% at the age of 6-10 months). Moreover, RetMEN2B/MEN2B animals presented with ganglioneuromas of the adrenal medulla (100% at 2-10 months) and

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enlargement of the associated sympathetic ganglia (Smith-Hicks et al. 2000). The described mouse model showed two of the main tumor features of the MEN2B syndrome: CCH and pheochromocytoma. Surprisingly, neither multiple mucosal neuromas nor ganglioneuromas

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of the gastrointestinal tract, tumors typically used to distinguish between MEN2A and MEN2B, could be found in either heterozygous or homozygous mutant mice. Taken together,

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the homozygous mice modeled the human MEN2B syndrome more closely than the heterozygous ones, in that they displayed an early incidence and increased severity of CCH and chromaffin cell hyperplasia, but differed from the human syndrome for two main aspects: they did not develop MTC (up to 12 months of age) or ganglioneuromas of the gastrointestinal tract. The majority of the available mouse models of oncogenic RET function were generated by tissue-specific expression (mainly in thyroid C-cells) of mutated RET alleles (Table 1).

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ACCEPTED MANUSCRIPT It has been demonstrated that alternative splicing of exons and introns between exons 19 and 21 gives rise to 3’ variants that encode for 3 human RET isoforms named RET9, RET 43 and RET51. These isoforms have a different number of amino acids at their C-terminus (Myers et al. 1995). Michiels and colleagues generated transgenic mouse lines expressing

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the RET9 isoform with the MEN2A-associated point mutation at codon 634 (C634R) (Michiels et al. 1997). In these animals, the expression of the mutated RET gene was targeted to the thyroid C-cells by placing it under the control of the CGRP/CT (calcitonin gene-related peptide / calcitonin) rat promoter. Mice expressing the transgene in the thyroid

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C-cells displayed overt bilateral CCH followed by the development of multifocal and bilateral MTC with complete penetrance at 14 months in two transgenic mouse lines, and with

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incomplete penetrance in one transgenic line. These tumors were morphologically and biologically similar to human MTC, demonstrating that the short isoform of RET is capable of driving tumorigenesis in vivo (Michiels et al. 1997). CT is the classical tumor marker of MTC, and evaluation of its levels in basal or stimulated conditions can be used for the diagnosis

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and follow-up of MTC. In predisposed MEN2 patients, the identification of CCH or MTC relies on the measurement of serum CT levels before and after stimulation with pentagastrin or calcium (Colombo et al. 2012). Following calcium stimulation, CT serum levels increased

pathology.

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also in the RET-MEN2A transgenic mice and this correlated with the presence of thyroid This observation is reminiscent of what happens in MEN2 patients bearing

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thyroid tumors, where abnormal calcitoninemia after calcium and/or pentagastrin provocative test is usually observed (DeLellis 1995). Although biochemical comparisons between the RET9 and the RET51 isoforms suggest differences in intracellular substrate binding capabilities (Lorenzo et al. 1997), in cellular transformation and differentiation activities (Marshall et al. 1997; Rossel et al. 1997), the functional significance of these in vitro observations on development and tumorigenesis in vivo remained unclear. Thus, Reynolds and colleagues (Reynolds et al. 2001) addressed this issue by generating a MEN2A mouse model in which the human RET51 cDNA, instead of 11

ACCEPTED MANUSCRIPT the shorter RET9 cDNA (Michiels et al. 1997), was expressed under the control of the human C-cell specific CT promoter (line CT-2A) (Reynolds et al. 2001). These mice developed CCH by 3 months of age (35% of cases), and then bilateral MTC by 6-12 months, with a frequency of 52%, which rised to 68% in mice over 12 months of age. This age-dependent tumor

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progression resembles the situation in MEN2 patients. Additionally, one founder line of these transgenic mice (line 2A-1) developed thyroid follicular tumors resembling human PTC (papillary thyroid carcinoma) with a penetrance of 50% (at 3-24 months of age) and exocrine pancreatic tumors (22%). These results were quite surprising as PTC is only rarely

pancreatic tumors are not seen in these patients.

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associated with the human MEN2A syndrome (Decker 1993; Oishi et al. 1995) and

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Other groups created tissue-specific transgenic mice expressing the human RET oncogene cDNA encoding the RET9 isoform in which the MEN2B-specific M918T mutation had been introduced (Sweetser et al. 1999; Acton et al. 2000). Acton and colleagues (Acton et al. 2000) used the human CT gene promoter (CALC-I) to generate transgenic mice (CALC-

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MEN2B-RET) expressing the human RET proto-oncogene with the above mentioned mutation exclusively in the thyroid C-cells. In three of eight CALC-MEN2B-RET transgenic founder lines, nodular CCH which progressed to MTC with age (frequency 37,5% at the age

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of 20 months) was observed (Acton et al. 2000). These transgenic mice showed a histopathological phenotype similar to MEN2B patients, with the initial appearance of hyperplastic

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nodular foci and the subsequent development of multifocal and bilateral MTC. As mentioned above, the measurements of basal plasma CT levels may give an indication for the presence of CCH or MTC in patients. Thus, Acton and colleagues monitored several CALC-MEN2BRET lines using this approach and could detect highly elevated plasma CT levels in mice harboring MTC. In two other studies (Gestblom et al. 1999; Sweetser et al. 1999), transgenic mice were generated in which the human DβH (dopamine β-hydroxylase) promoter was used to target expression of the MEN2B-specific M918T mutation selectively in sympathetic neurons, 12

ACCEPTED MANUSCRIPT adrenal chromaffin cells, enteric neurons and their neural crest-derived precursors (Kapur et al. 1991; Mercer et al. 1991). Mice expressing this transgene (DβH-RETMEN2B) developed benign neuroglial hyperplasia in their sympathetic ganglia (100% penetrance) and in the adrenal glands, which were histologically identical to the ganglioneuromas observed in patients

but

failed

to

develop

mucosal

ganglioneuromas

or

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MEN2B-affected

pheochromocytomas.

Altogether, these different transgenic mouse models provided compelling evidence that the mutated Ret/RET alleles are oncogenic in parafollicular C-cells and suggested that these

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mice constitute a valuable tool to investigate the role of RET in the pathophysiology of human MTC (Table 1). Moreover, they offer the possibility to study the early stages of tumor

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formation (i.e. CCH), something that is very difficult to perform in human patients, and may be useful to evaluate new diagnostic methods, and to test novel drugs which might inhibit RET function and impair tumor growth.

The rat MENX syndrome as a model of MEN4

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Over 10 years ago, a Sprague-Dawley (SD)-derived rat cohort bred at our Institute started to spontaneously develop multiple endocrine tumors with high penetrance. Histo-pathological examination revealed that the tumors developing in these rats overlap the tumor spectrum of

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both MEN1 and MEN2 human tumor syndromes (Fritz et al. 2002). Specifically, affected rats presented with anterior pituitary adenoma, bilateral adrenal pheochromocytoma as well as

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extra-adrenal pheochromocytoma (paraganglioma), thyroid C-cell hyperplasia, parathyroid hyperplasia and pancreatic islet cells hyperplasia (Table 1). This multiple endocrine tumor syndrome was named MENX (Fritz et al. 2002). In contrast to the human MEN syndromes, MENX was inherited as a recessive trait, suggesting that affected rats are homozygous for the underlying genetic mutation. Mutant rats weighted more than their wild-type littermates and, in addition to the multiple tumors, they showed organomegaly, particularly of spleen and thymus, and juvenile cataracts (Pellegata et al. 2006). The average life span of affected rats

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ACCEPTED MANUSCRIPT was 10±2 months whereas their wild-type littermates lived approximately 24-30 months (Pellegata et al. 2006). In an attempt to identify the genetic mutation underlying MENX, linkage studies were performed and the MENX locus was initially mapped to an interval of approximately 22 cM on

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the distal part of rat chromosome 4 (Piotrowska et al. 2004). On this chromosomal region maps the rat homologue of the RET gene, which was however subsequently excluded as being the candidate gene for the MENX syndrome (Piotrowska et al. 2004). When the MENX locus was fine-mapped to a ~3 Mb interval, suitable candidate genes were identified and

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sequenced. Among them was Cdkn1b, encoding the cyclin-dependent kinase (CDK) inhibitor p27 (Figure 1), which was selected because p27-/- mice develop pituitary adenomas (a tissue

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also affected in MENX mutant rats) and because p27 was considered a putative tumor suppressor, consistent with the recessive mode of inheritance of the rat syndrome. Upon sequencing the Cdkn1b gene in affected and unaffected littermates, a tandem duplication of 8 nucleotides in exon 2 (c. 520-528dupTTCAGAC), which causes a frameshift, was identified

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in affected rats. At the protein level the mutated allele encodes a protein predicted to have a novel C-terminal sequence starting at codon 177 and to be 23 amino acids longer than the wild-type p27 protein. Whereas tissues of affected rats were found to express the mutant

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Cdkn1b transcript at a level similar to the wild-type mRNA, they displayed extremely reduced or absent p27 expression following immunohistochemical staining with a specific anti-p27

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antibody (Pellegata et al. 2006). Subsequent in vitro studies demonstrated that the MENXassociated mutant p27 protein is very unstable and rapidly degraded, in part, by the proteasome (Molatore et al. 2010a). In the past few years, we have endeavored to characterize the tumors developing in MENX rats at different levels (histo-pathological, molecular, ultrastructural, physiological) to assess the potential similarities with the corresponding human tumors. The first pathological alterations observed in mutant rats affect the adrenal medulla: they presented with adrenomedullary hyperplasia at around 4 months of age (frequency 100%), 14

ACCEPTED MANUSCRIPT which invariably progressed to pheochromocytoma by 6-8 months. The tumors were histologically similar to human pheochromocytoma: the neoplastic medullary cells were arranged into small nests or alveoli (“Zellballen”) with a rich vascular network, and the large tumors displaced the surrounding normal cortex (Shyla et al. 2010). The rat adrenal

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chromaffin cells are divided into two sub-populations based on catecholamine biosynthetic enzyme expression and the type of hormone released, i.e. adrenergic and noradrenergic chromaffin cells. These two cell types can be distinguished based on the expression of the enzyme

phenylethanolamine

N-methyltransferase

(PNMT),

which

catalyzes

the

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transformation of noradrenalin into adrenalin and is present in adrenergic chromaffin cells. We observed that, during tumor progression, the chromaffin cell population that expanded in

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the adrenal of MENX rats was composed of PNMT-negative cells, suggesting that the tumors derive from noradrenergic cells (Figure 2). The proliferation rate of rat pheochromocytomas, measured as Ki67 labelling index, was variable from lesion to lesion and was on average 11.3% (range 3.7% and 16.7%) (Miederer et al. 2011), higher than the typical human tumors

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and likely due to the lack of p27-dependent cell cycle control. Despite these high proliferation rates, no metastases of rat pheochromocytomas have been so far documented. At the molecular level, a genome-wide scan for allelic imbalance (AI) in rat

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pheochromocytomas identified recurrent allelic losses at candidate regions on rat chromosomes 8 and 19 (Shyla et al. 2010). Interestingly, the regions often lost in rat tumors

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were syntenic to regions previously shown to be involved in human pheochromocytomas. Thus, studies of the genomic alterations in rat tumors might facilitate the identification of novel candidate genes implicated in their human counterpart. Gene

expression

array

analyses

of

rat

adrenomedullary

hyperplasia

and

pheochromocytomas were also performed, and have demonstrated that the rat lesions share molecular pathways with the human tumors and can therefore be exploited as a gene discovery platform. Genes were identified that are overexpressed in both rat and human pheochromocytoma and represent novel tumor markers of these tumors (i.e. BMP7, 15

ACCEPTED MANUSCRIPT PHOX2A and other developmental genes) (Molatore et al. 2010b). The similarities between rat and human adrenomedullary tumors also extend to the uptake of radiolabeled tracers for scintigraphy (Single Photon Emission Computed Tomography, SPECT) or Positron Emitted Tomography (PET). In patients with pheochromocytoma, functional imaging can be

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performed using radioiodinated metaiodobenzylguanidine (MIBG) for planar scintigraphy and SPECT with high sensitivity and specificity (Havekes et al. 2008). Tumor uptake of MIBG is mediated by the norepinephrine transporter (NET), which is abundant on the surface of pheochromocytoma cells (Havekes et al. 2008).

18

F-L-6-fluoro-3,4-dihydroxyphenylalanine

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(18F-DOPA), targeting the aromatic amino acid transporter and L-amino acid decarboxylase (Hoegerle et al. 2002; Havekes et al. 2008; Martiniova et al. 2012), and 68

Ga-DOTATOCand

68

Ga-labeled

Ga-DOTATATE), targeting somatostatin

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somatostatin analogs (e.g.,

68

receptors (Win et al. 2007), can also be used to visualize pheochromocytomas in man. We could demonstrate that the rats tumors can be noninvasively visualized using (Gaertner et al. 2013),

68

Ga-DOTATOC and

analog,

18

18

I-MIBG

11

C- Hydroxyephedrine (HED; a norepinephrine

F-DOPA (unpublished), as well as a novel norepinephrine

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analog) (Miederer et al. 2011),

131

F-LMI1195 (Gaertner et al. 2013). For all these tracers, higher uptake was

observed in mutant rats bearing adrenomedullary tumors compared to wild-type adrenal

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glands.

Pituitary tumors are the second lesion that temporally occurs in mutant rats. The initial

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lesions in the pituitary gland of MENX mutants appeared at about 4 months of age and histologically presented as distinct, discrete nodules. At 5 months, lesions started to merge and by the age of 8 months, they appeared as large adenomas that almost completely effaced the gland (Marinoni et al. 2013). Adenomas at 8 months exhibited significant increase in the number of blood vessels in comparison with early lesions. Although these adenomas reached a considerable size, no invasion of the skull base or brain tissue was observed and none of the lesions metastasized within or outside the cranio-spinal axis. The lesions in rats at 4-6 months of age showed variable expression of the luteinizing hormone 16

ACCEPTED MANUSCRIPT beta subunit (LHβ) and the follicle stimulating hormone beta subunit (FSHβ) and widespread expression of alpha subunit (αGSU), the common subunit of the gonadotropin hormones (Figure 3). Expression of the other pituitary hormones was rarely seen in the rat adenomas. The number of cells expressing LHβ or FSHβ decreased with the increase in size of the

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nodules and became negligible in the largest adenomas, while tumor cells remained positive for αGSU all throughout tumor progression (Figure 3). Decreased LHβ expression over time reflected in reduced serum levels of this hormones in adult female and male mutants rats

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bearing pituitary adenomas compared to sex- and age-matched wild-type rats (Marinoni et al. 2013). All adenoma cells featured nuclear immunoreactivity for the transcription factor SF-1,

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typical of gonadotroph cells. Altogether, these findings demonstrated that the rat pituitary tumors are gonadotroph adenomas (Figure 3). Given that the proliferation rates are quite elevated, reaching an average Ki67 labelling index of 8% at 8 months (range from 1% to 21%) (Marinoni et al. 2013), the rat adenomas better resemble human aggressive gonadotroph adenomas.

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To discover new genes/pathways involved in gonadotroph cells tumorigenesis, we performed transcriptome profiling of rat tumors versus normal rat pituitary. Rat adenomas showed overrepresentation

of

genes

involved

in

cell

cycle,

development,

cell

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differentiation/proliferation, lipid metabolism (Lee et al. 2013) (Figure 4). Meta-analyses demonstrated remarkable similarities between gonadotroph adenomas in rats and humans,

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and identified common dysregulated genes not previously implicated in pituitary tumorigenesis. Two such genes, CYP11A1 (encoding the steroidogenic enzyme P450cc) and NUSAP1 (encoding a protein important in mitotic spindle assembly), were found to be up-regulated in 77% and 95%, respectively, of human gonadotroph adenomas. Gene overexpression translated into high levels of the P450scc and NuSAP proteins in the human adenomas (Lee et al. 2013). These studies revealed clues to the molecular mechanisms driving rat and human gonadotroph adenoma development, identified previously unexplored

17

ACCEPTED MANUSCRIPT biomarkers and established similarities in gene expression between rat and human pituitary adenomas. Given their close resemblance to the human adenomas, the rat neoplasms are suitable for testing novel drugs for their efficacy against gonadotroph adenomas in vitro (using primary

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tumor cells) (Lee et al. 2011) and in vivo (in MENX rats) (Lee et al. 2015). In a recent preclinical study, we evaluated the response of rat pituitary adenomas to a novel dual inhibitor of the PI3K/mTOR signaling cascade (Lee et al. 2015). This pathway is hyperactivated in both rat and human gonadotroph adenomas. We could show that this drug

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(BEZ235) elicits a potent anti-tumor response in the rat tumors, which can be monitored by functional imaging using diffusion weighted magnetic resonance. These findings provided a

adenomas (Lee et al. 2015).

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Conclusion

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rationale for the clinical investigation of PI3K/mTOR inhibition in human gonadotroph

Animal models of syndromic cancers have sometimes been disappointing as they could not always recapitulate the human disease they were meant to model (i.e. mice defective in Brca1 do not develop mammary cancer). This is not the case for the mouse models of MEN1

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and MEN2 syndromes here presented. Indeed, these genetically engineered mice were

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shown to quite closely reflect the pathology and physiology of the human syndromes. Although the various lines generated have some phenotypic differences with the corresponding human disease, they significantly contributed to our knowledge of the role of the predisposing genes in endocrine tumorigenesis, and provided formal proof of their tumor suppressive or oncogenic role in these cells. The rat MENX syndrome has provided us with a novel tumor susceptibility gene for multiple neuroendocrine tumors in both rats and humans: Cdkn1b/CDKN1B (p27). The similarities between the rat tumors and their corresponding human tumors at various levels suggest that 18

ACCEPTED MANUSCRIPT MENX-associated neoplasia can be exploited to elucidate the molecular mechanisms driving tumorigenesis in their corresponding human tumors and to perform preclinical trials

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evaluating novel targeted therapies against these tumors.

ACKNOWLEDGMENTS

We thank the members of the laboratory for stimulating discussions. The work of the authors

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is supported by the Deutsche Forschungsgemeinschaft SFB824, subproject B08, and by the

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Deutsche Krebshilfe (grant 109223).

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ACCEPTED MANUSCRIPT Figure Legends Figure 1. Schematic representation of the role of p27 as cyclin-dependent kinase (CDK) inhibitor with a special emphasis on the G1-S phase transition. The binding of p27 to CyclinD/CDK4,6 complexes facilitates their import into the nucleus without inhibiting their

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kinase activity. Upon mitogenic stimulation, CyclinD/CDK4,6 complexes start phosphorylating the Rb protein in mid G1. Phosphorylated Rb releases the transcription factor E2F which induces the expression of genes required for the G1 to S progression, including Cyclin E.

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CyclinE/CDK2 complexes phosphorylate p27 on Thr187 and this generates a signal for proteasome degradation, so that, following the removal of p27, CDK2 can further

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phosphorylate Rb and release E2F factors.

Figure 2. Pheochromocytoma cells in MENX rats do not express PNMT. (A) Histological examination by H&E staining of the adrenal gland of a MENX mutant rat at 8 months of age. Size bar: 500 µm. (B) Magnified view of the adrenal gland shown in A. Size bar: 200 µm. (C)

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Further enlargement of one part of the adrenal gland shown in A. Size bar: 100 µm. (D) Immunohistochemical staining for PNMT of a section next to that shown in C. The PNMTpositive cells are the residual normal adrenomedullary cells displaced by the growing tumor.

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Size bar: 100 µm. T, tumor area.

Figure 3. Characterization of the pituitary adenomas in MENX rats. Sections of normal

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anterior pituitary glands in wild-type (WT) rats and sections of the pituitary adenomas of mutant rats are shown. For the tumors, only the tumor area is here illustrated. Immunohistochemical staining was performed using specific antibodies against the indicated proteins. While immunoreactivity for LHβ and FSHβ decreases with tumor progession in the tumors of mutant rats, αGSU expression is maintained. SF-1 positivity attests to the gonadotroph origin of these tumors, which also show quite elevated proliferation rates, as assessed by Ki67 staining. Size bar: 20 µm.

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ACCEPTED MANUSCRIPT Figure 4. Microarray analysis of rat pituitary adenomas and control tissues. (A) Heat map of the probe set IDs significantly dysregulated in the pituitary tumors of mutant rats (MUT) compared with pituitary tissues of wild-type age-matched rats (WT). Samples were ordered by hierarchical clustering. Red (blue) indicates higher (lower) expression level with respect to

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the median across all samples in each group. On the right are listed some of the dyregulated genes and the Gene Ontology category to which they belong. The log2 scale is provided at the bottom. (B) Graphic representation of the percentage of the most differentially expressed

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belong. The names of the categories are listed below.

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genes in tumors versus control pituitary and of the different GO categories to which they

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Table 1. Animal models of multiple endocrine neoplasia type 1 (MEN1), type 2 (MEN2) and of MENX presented in this review.

Conventional 3–8

Conventional 3

Conventional 2

Conventional 1–2

NIH Black Swiss, Mixed 129/Ola x 129/Sv 129/SvEvTacFBR C57BL/6_129

Mixed C57BL/6_129

Background strain

C57BL/6J

Outcome

Embryonic lethality

Viable

Viable

Viable

Viable

Viable

Viable

Viable

Viable

Parathyroid

−

+ (24%)

+ (41%)

+ (17%)

−

−

+ (↑Ca) (80%)

−

Pancreas

−

+ (INS 28%)

+ (INS, GLU) (65%)

+ (INS, GLU) (90%)

+ (INS)

+ (INS)

−

−

Pituitary

−

+ (PRL 26%)

+ (PRL, GH) (19 - 37%)

+ (PRL, NF) (F:70%; M:30%)

−

−

−

−

Adrenal cortex

−

+ (20%)

+ (46%)

+ (8%)

−

−

−

−

Phaeochromocytoma

−

+

−

+

+ (INS, GLU, SSTR2, CgA) (60%) + (PRL, GH, ACTH, SSTR2, CgA) + (3β-HSD), ↑Cort + (TH)

−

−

−

−

Gastric NET

−

+

+

−

−

−

−

−

(+)

+ (↑Ca)

TE D

M AN U

Tumour spectrum:

Conditional Conditional 3–8 3 Mixed Mixed C57BL/6_FVB_1 C57BL/6_129/Sv 29/Sv

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Men1 exons deleted

Conventional 2–4

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Models of MEN1 Type:

Conditional 3–8 Mixed FVB_129/Sv

Conditional 3–8 Mixed C57BL/6J_FVB_1 29/S

−

+

+ (6,5%)

+ (20%)

+ (11,5%)

−

−

−

−

−

−

+ (88%)

+ (30%)

+

−

−

−

−

Extra pancreatic gastrinoma

−

−

+

−

−

−

−

−

−

Ovarian (sex-cord)

− Scacheri et al. 2001

+ Crabtree et al. 2001

+ (50%) Bertolino et al. 2003a

+ (16%) Loffler et al. 2007

+ Harding et al. 2009

− Crabtree et al. 2003

− Bertolino et al. 2003b

− Libutti et al. 2003

− Veniaminova et al. 2004

Conditional

Conditional

Conditional

Conditional

Conditional

C634R

C634R

M918T

M918T

M918T

hum DβH

hum DβH

Models of MEN2

Conventional

Type Men2 mutation

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References

M919T (Heterozygous)

M919T (Homozygous)

---

---

RET isoform Promoter

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Thyroid Testicular (Leydig-cell)

RET9

RET51

RET9

CT

hum CT

hum CT

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Background strain

C57BL/6J

C57BL/6J_DBA2

Outcome

Viable

Viable

FVB_C57BL/6J_ C57BL/6J_DBA2 CBA Viable Viable

C57BL/6J_SJL Viable

DBA_C57B1/6_ C57BL/6J Viable

Tumour spectrum: + (CCH)

(41%) + (CCH)

(86%)

+ (CCH) (35%); + (CCH) (MTC) (MTC) (68%); (37,5%) ↑CT in (PTC) (50%) MTC

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Thyroid

+ (CCH, MTC) ↑CT

−

−

−

+ (100%)

−

−

−

+

+

Pancreas

−

−

−

+ (exocrine)

−

−

−

References

+ (18%) + (100%) Smith-Hicks et al. 2000

− Michiels et al. 1997

MENX Spontaneous

Cdkn1b mutation

c.520–528dupTTCAGAC (frameshift)

Strain

Sprague-Dawley

Outcome

Viable

Tumour spectrum Parathyroid

+

Pancreas

+

Pituitary

+ (100%)

Multifocal

Phaeochromocytoma

+ (100%)

Bilateral

References

+ Medullary Molatore and Pellegata 2010c

(LH, FSH, αGSU)

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Thyroid

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Type

− − − Reynolds et al. Acton et al. 2000 Gestblom et al. 2001 1999

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Adrenal medulla

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Ganglioneuromas

Marinoni et al. 2013

− Sweetser et al. 1999

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EP

TE D

M AN U

SC

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AC C

EP

TE D

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SC

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AC C

EP

TE D

M AN U

SC

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SC

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ACCEPTED MANUSCRIPT Animal models of MEN1

•

Animal models of MEN2A and MEN2B

•

The rat MENX syndrome as a model of MEN4

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•